We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a G protein and during the course of translation initiation the GTP is hydrolyzed to GDP. eIF2 is released from the ribosome in complex with GDP and the guanine-nucleotide exchange factor eIF2B converts eIF2-GDP to eIF2-GTP. Phosphorylation of the alpha subunit of eIF2 on serine 51 coverts eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA to down-regulate protein synthesis in virally infected cells), PERK (activated under conditions of ER stress), and HRI (activated under conditions of low heme). eIF2 is composed of three subunits. The gamma subunit of eIF2 is a GTPase that resembles elongation factor EF-Tu. We previously showed that despite their structural similarity eIF2 and EF-Tu bind tRNA in substantially different manners, and we showed that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. As described below, our structure-function studies on eIF2 have provided insights into human disease. Whereas protein synthesis plays a critical role in learning and memory in model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Working with collaborators in Israel and Germany, we characterized a human X-linked disorder characterized by intellectual disability and microcephaly. The patients carry a mutation in the EIF2S3 gene encoding eIF2gamma, and genetic and biochemical studies revealed that the mutation disrupts eIF2 complex integrity and translation start codon selection. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function. Over the past year, working with additional collaborators, we have are characterizing new mutations in eIF2gamma that cause intellectual disability. When expressed in yeast, human PKR phosphorylates the alpha subunit of eIF2 on Ser51 causing inhibition of protein synthesis and yeast cell growth. To subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. The poxviral protein E3L binds double-stranded RNA inhibits PKR by sequestering activators and forming heterodimers with the kinase. We previously showed that a Z-DNA binding domain near the N-terminus of E3L, but not its Z-DNA binding activity, was critical for E3L inhibition of PKR. We are currently characterizing mutations in PKR that confer resistance to E3L inhibition. We previously discovered that the insect baculovirus PK2 protein is an eIF2alpha kinase inhibitor. PK2 structurally mimics the C-terminal lobe of a protein kinase domain. Using a genetic screen in yeast, and together with collaborators in Canada and Japan, we characterized mutations that enhance the ability of PK2 to inhibit eIF2 kinases. These mutations cluster to a surface of PK2 that in bona fide protein kinases forms the catalytic cleft through interactions with a kinase N-lobe. Yeast two-hybrid and protein interaction assays revealed that PK2 associates with the N-lobe of PKR. Using yeast-based assays, we showed that PK2 was most effective at inhibiting an insect HRI-like kinase, and our collaborators showed that knockdown of the HRI-like kinase in insects rescued viral defects associated with loss of PK2. We propose an inhibitory mechanism whereby PK2 engages the N-lobe of an eIF2&#945; kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Whereas GCN2, HRI, PKR, and PERK specifically phosphorylate eIF2alpha on Ser51 to regulate translation, eIF2alpha is dephosphorylated by the broadly acting serine/threonine protein phosphatase 1 (PP1). In mammalian cells, the regulatory subunits GADD34 and CReP target PP1 to dephosphorylate eIF2alpha; however, there are no homologs of these targeting subunits in yeast. We previously showed that a PP1-binding motif present on an N-terminal extension that is unique to yeast eIF2gamma directs the yeast PP1 (GLC7) to dephosphorylate eIF2alpha. Over the last year we have continued our studies on eIF2alpha dephosphorylation and we have reconstituted human GADD34 function in yeast cells. We mapped a novel eIF2&#945;-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. Point mutations altering the 19-residue eIF2&#945;-binding motif impaired the ability of GADD34 to interact with eIF2&#945;, promote eIF2&#945; dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, the eIF2&#945;-docking motif is conserved among viral orthologs of GADD34, and we showed that it is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2&#945; dephosphorylation. Taken together, our data demonstrate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2&#945; by interacting with both PP1 and eIF2&#945; through independent binding motifs. We are also studying the translation factor eIF5A. eIF5A is the sole protein containing the unusual amino acid hypusine, N-epsilon-(4-amino-2-hydroxybutyl)lysine. The hypusine residue in eIF5A is found in all eukaryotes and archaea. Using molecular genetic and biochemical studies, we showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. More recently, we showed that eIF5A from yeast, like its bacterial ortholog EF-P, stimulates the synthesis of proteins containing runs of consecutive proline residues. Consistent with these in vivo findings, we showed that eIF5A was critical for the synthesis of polyproline peptides in reconstituted yeast in vitro translation assays. Using directed hydroxyl radical probing experiments, we mapped eIF5A binding near the E site of the ribosome with the hypusine residue of eIF5A adjacent to the acceptor stem of the P-site tRNA. Thus, we propose that eIF5A, like EF-P, stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor substrates like proline. In addition to studying translation factors and regulators, we have also studied the impact of mRNA structure on translation. The HAC mRNA in yeast encodes a transcription factor that up-regulates genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the leader of the HAC1 mRNA represses Hac1 protein production under basal conditions. An unusual cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under conditions of ER stress, relieves the inhibition and enables Hac1 synthesis. Using random and site-directed mutations we showed that disruption of the base-pairing interactions derepresses translation of the unspliced HAC1 mRNA. With our collaborators we also showed that insertion of an in-frame AUG start codon upstream of the base-pairing interaction releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhanced production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. As the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes, we conclude that the 5' UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA.